Effect of Blood Flow Restriction on Serum Brain-Derived Neurotrophic Factor
ABSTRACT
Background
Blood flow restriction exercise (BFRE) is a therapeutic approach commonly used to facilitate muscular strength and hypertrophy. Emerging evidence has identified its benefits on other systems and metabolic processes. The purpose of this study was to examine potential impact of BFRE on serum levels of brain-derived neurotrophic factor (BDNF).
Methods
Eighteen healthy adults (9 men, 9 women; mean age 34.44 ± 9.97 years) were randomized into groups to perform cycling either with or without blood flow restriction (BFR). Blood samples were collected before and after exercise to analyze serum concentrations of BDNF using enzyme-linked immunosorbent assay.
Results
Between groups analysis of variance found BDNF significantly higher postexercise in the BFR group. There was a main effect for sex indicating women using BFR with exercise had a greater increase in BDNF than men (P < 0.05).
Conclusion
Cycling with BFR may induce significant increases in serum levels of BDNF. Further research should continue examining the impact of BFRE on serum levels of BDNF using this methodology in other populations to increase generalizability of results and explore its use in the prevention of age-related cognitive decline. In addition, a longitudinal design would investigate the impact of time.
INTRODUCTION
Blood flow restriction exercise (BFRE), also known as vascular occlusion training, is a therapeutic approach used to facilitate muscular strength and hypertrophy in individuals who are unable to tolerate heavy loads (1). Although foundational studies have examined the utility of BFRE in rehabilitation of orthopedic injuries and general deconditioning, recent evidence supports novel utilization of BFRE to alter serum concentration of an analyte important in brain health (1–3).
Evidence has emerged emphasizing the existence of a bidirectional relationship between physical activity and cognitive function (4). A deeper understanding of how exercise induces changes in neurobiological mechanisms impacting cognition is needed to develop efficient strategies to combat age-related cognitive decline (5). Brain-derived neurotrophic factor (BDNF) facilitates cognitive performance because of enhanced neuroplasticity (6,7). This study investigates if BFRE can increase serum levels of BDNF compared with controls. If this is demonstrated, it could be promising as a physical activity strategy to mitigate age-related cognitive decline.
A study in rats indicated that BDNF levels in both plasma and serum correlate with levels of the protein in the brain (8). Several nonhuman studies corroborated that circulating levels of BDNF can serve as a proxy for expression in the brain (9,10).
Moreover, lactate is associated with changes in peripheral BDNF (3,9). El Hayek et al. (11) identified that lactate can improve cognitive function by promoting hippocampal-dependent learning and memory in a BDNF-dependent manner via tropomyosin receptor kinase B signaling. The action of lactate is dependent on the activation of sirtuin 1 deacetylase, which increases levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha. This increase, in turn, coordinates the increase in fibronectin type 3 domain containing 5 expression, which is known to induce BDNF expression (11). Individuals who have undergone BFRE have demonstrated a significant rise in blood lactate levels when compared with work-matched controls without blood flow restriction (BFR) and comparable with those participating in high-intensity exercise (12). To isolate the effect of lactate on BDNF, Schiffer et al. (13) performed an experiment using the lactate clamp method at rest to determine the role of lactate in regulation of blood concentrations of BDNF. Eight healthy male athletes (average age 25 years) received 4.2 mmol of sodium lactate in an incremental design. Venous blood samples were taken before and after the infusion as well as 24 and 60 min postinfusion. The blood samples were centrifuged and stored at −40°C until analysis of serum BDNF. The rise of lactate was accompanied by a significant increase of serum BDNF (P < 0.05) in a dose-dependent fashion (13).
Although studies have shown that physical activity increases levels of BDNF (14), further research is needed to identify specific methods of physical activity that will foster the strongest results. Recent studies have found BFRE may increase serum levels of BDNF (15,16).
A study by Du et al. (15) assessed BDNF along with lactate in 24 participants (average age 47.83 ± 4.83) with poststroke depression. Each participant underwent 3 randomized resistance exercise tests: low intensity (40% of 1-repetition maximum [1 RM]), BFRE (40% 1 RM), and high intensity (80% 1 RM) with 3 d between each test. All groups performed 3 sets of 10 repetitions of the following exercises: seated pull-down, seated shoulder press, seated chest press, seated chest push, and seated leg kick, with 120-second resting periods between sets. Analysis between groups found that changes in blood lactate and BDNF levels in the BFRE and high-intensity groups were significantly higher than those in the low-intensity group (P < 0.05). The authors reported no significant difference between the BFRE group and the high-intensity group (P > 0.05). Authors concluded that BFRE may increase serum BDNF levels in individuals with poststroke depression by increasing blood lactate concentration (15).
Kargaran et al. (16) performed a study examining the impact of dual-task training with and without BFR on cognitive function in 24 healthy older women (age 62.9 ± 3.1 years). Participants were randomly assigned into either dual task, dual task with BFR (DTBFR), or a control group. The experimental groups performed cognitive tasks while walking on a treadmill at 45% of heart rate reserve (20 min per session, 3 sessions per week for 8 weeks). Results reveals plasma BDNF concentration in both experimental group, DTBFR (P < 0.001) and dual task (P < 0.005), were significantly greater than the control group. The changes in BDNF in response to the interventions were also found to be positively correlated with performance on the Mini Mental State Examination (16). Although it is possible that the increase of BDNF in the DTBFR group could be due to the dual task, the positive impact on cognitive performance with a prospective design adds value to the results.
These studies suggests that high-intensity exercise at 80% of 1 RM, BFRE with low-intensity 45% of heart rate reserve, and resistance training 40% of 1 RM are optimal approaches to generate enough lactate to increase circulating BDNF when compared with low-load or low-intensity exercise alone (15,16). It is essential to have an alternative approach to high-intensity exercise because there are demographics, such as the older population, with joint or cardiovascular pathologies that are unable to tolerate high loads or intensities.
The current study aimed to explore the use of BFR during an aerobic cycling exercise with upper and lower extremities on serum levels of BDNF. The investigators hypothesized that the mean values for BDNF would increase following BFRE when compared with a control group.
METHODS
Participants
University of Central Arkansas Institutional Review Board approval was attained on May 4, 2023 (IRB#23-099). Eighteen healthy participants, ages 18 to 50 years (mean age 34.44 ± 9.97 years), were enrolled in the study. This sample size was derived from a priori G*power calculations using a moderate to large effect size (F = 0.75). To obtain baseline data from a normative population, healthy adults with no history of previous chronic illness and with no acute illness within the last 3 months were recruited to participate. Participants were excluded if they had a resting systolic blood pressure exceeding 140 mm Hg or a diastolic blood pressure exceeding 90 mm Hg at the time of study participation, currently taking antihypertensive drugs, a body mass index ≥30, any acute orthopedic injury, an ankle-brachial index outside 0.90 to 1.26, neurologic or cardiovascular diseases that would limit safe exercise, a personal or family history of blood clotting disorder, or reported smoking in the last 6 months.
Procedures
Data collection times were scheduled between 8:00 and 10:00 AM for all participants to minimize diurnal variation in blood analytes. The participants were asked to fast for 12 h and avoid alcohol for 24 h before data collection to minimize variation in analytes.
To ensure equal representation of men and women in each group, a stratified block randomization of participants was utilized. For example, when a male participant was enrolled, he was first allocated to the male stratum, then group allocation (BFRE or controls without BFR) was determined through block randomization applied to the male stratum. The same assignment method was used for female participants. The blocks of 4 were computer generated using an Excel random number generator.
Upon arrival at the data collection site, participants were instructed to rest in a seated position for 10 min to diminish postural effects on blood analytes. Following the allotted resting time, each participant’s blood pressure was assessed to ensure it was within inclusion limits. If blood pressure was outside of inclusion limits, the participant rested for an additional 10 min and was reassessed. If the average of the 2 blood pressures did not meet inclusionary requirements, the participant was excluded from the study. Additionally, each participant’s ankle-brachial index was assessed in supine to screen peripheral artery function. If the ankle brachial index measured outside of the normative range for healthy adults (17), the participant was excluded.
Venous blood samples were obtained from the left antecubital vein immediately before and after exercise to assess changes in serum BDNF levels in the blood following exercise. Blood samples were drawn by a licensed, registered nurse and collected in red-top vials that promote blood coagulation. Before exercise, 1 vial of blood was obtained from a single blood draw. Upon sampling, vials were inverted to mix and placed on ice until processing via centrifuge.
Following the initial blood draw, all participants began cycling on an Assault bike (Assault Fitness, Carlsbad, CA; high-tensile steel), which is a low-impact, high-output machine that combines the arm action of an elliptical trainer with the lower body and cardiovascular workout of a stationary bike. During the exercise, both lower extremities and the upper extremity not utilized for the blood draw were in motion. The upper extremity from which the blood draw was obtained rested comfortably throughout the exercise. Participants were instructed to maintain a cycling intensity between 150 and 175 W with 45 to 50 rpm for 15 min, which mirrors the intensity and duration outlined in a previous BFR cycling study and a published case report of the current lead investigator (18,19). Physiologic exercise intensity was monitored using the Borg rating of perceived exertion (RPE) scale and heart rate response. The target RPE was 11, which presents as minimal sweating, light intensity, and ability to talk easily (20). To assess heart rate response to exercise, each participant wore a finger heart rate monitor on the resting upper extremity. The target heart rate response was established as 63% of the participant’s age-predicted heart rate maximum (220 minus age) plus or minus 10 beats per minute. This designated heart rate range correlates well with steady-state, low-intensity exercise at approximately 40% of maximum oxygen consumption (21).
During exercise, the investigator monitored RPE (via the participant’s ability to talk comfortably) and heart rate response. If targeted intensity parameters were exceeded, the cycling intensity was reduced until the participant regained ability to maintain the targeted RPE and heart rate values.
Participants assigned to the BFRE group wore elastic, pneumatically controlled BFR cuffs (BStrong, Park City, Utah; 5 cm wide × 50 cm long) around the upper arm on the arm opposite the blood draw (cuff pressure 160 mm Hg) and around the upper thigh of both lower extremities (cuff pressure 300 mm Hg). The lower extremity cuff pressures were determined based on a previous report using BStrong elastic cuffs (22) and the upper extremity cuff pressures mirrored a case study published by the lead investigator (18). After BFR cuffs were applied, but prior to initiating exercise, Doppler ultrasound was performed at the posterior tibial arteries at the medial ankle and radial artery at the anterolateral aspect of the wrist to ensure arterial inflow to the extremities. The BFR cuffs were monitored during each exercise session for consistency and safety by a licensed physical therapist who is certified in BFRE.
Participants assigned to the work-matched control group performed the same cycling exercise on the Assault bike at the previously described intensity without the BFR cuffs.
Blood Serum Analysis
Immediately following exercise, a registered nurse performed a second blood draw from the same antecubital vein used for the initial draw to extract serum for BDNF analysis. Once the postexercise blood draw was completed, blood samples sat for 30 min to allow for coagulation. At this point, samples were processed via centrifuge at 2,300 to 2,800 rpm for 10 min, allowing serum and plasma to be separated. The serum was aliquoted off the blood cells using a pipette, transferred to an Eppendorf tube, and immediately put on ice until they could be stored in a freezer at −20°C until enzyme-linked immunosorbent assay (ELISA) analysis was performed. The day ELISA was performed, the samples were taken out of the freezer and thawed at room temperature for 30 min prior to analysis.
Brain-derived neurotrophic factor analytes were measured using single-wash (simple step) colorimetric sandwich ELISA assay kits using an Abcam ELISA microplate reader. Each sample was added to well-plate strips that were precoated with immobilization antibodies. A capture and detection antibody cocktail was added to the sample, which was then incubated for 1 h. The well was washed, and a detection reagent was added to develop in proportion to bound complexes. After a 10-min incubation period, a stop solution was added to the well to prepare for quantification using a microplate reader.
To refine protocol logistics and reduce risk of procedural errors, a preliminary analysis was conducted with blood samples from 2 individuals who were not included in the formal study. Additionally, this preliminary analysis aimed to determine if the immunoassay was performing as expected and to determine if the investigator was able to achieve results in a reliable manner consistent with those established by experts. Procedures for blood sampling and processing for the preliminary analysis were performed consistent with methodology described for experimental study participants.
To establish procedural reliability, Abcam recommends creating and analyzing duplicates of standard concentration solutions and participant samples to provide adequate data for statistical validation of results. Once the standard concentration duplicates are developed, the average absorbance values for each set of standard solutions and participant samples can be obtained and used to calculate analyte concentration in units of picograms per milliliter.
For the preliminary analysis, a stock solution of BDNF was reconstituted with normal saline to yield a 2,000 pg·mL−1 solution. Fifty microliters of the reconstituted BDNF was pipetted into each well of the microplate to create standard concentration solution duplicates. Serial dilutions were performed on the stock solution of BDNF to identify optical density of the standard samples and duplicates. One hundred fifty microliters of normal saline was added to all standard control and duplicate wells. Next, 150 μL of BDNF stock solution was added to the first well, resulting in a 500 pg·mL−1 concentration of BDNF. Then, a systematic method of serial dilution was performed; 150 μL of the 500 pg·mL−1 concentration from well 1 was transferred to well 2 containing 150 μL of normal saline, yielding a 2-fold dilution to 250 pg·mL−1. Next, 150 μL of the 250 pg·mL−1 concentration from well 2 was transferred to well 3, yielding a 2-fold dilution at 125 pg·mL−1. The serial dilution continued until no BDNF was present. These standards served as a positive control with known concentrations of BDNF in pg·mL−1.
To identify the concentration of BDNF in the samples obtained from preliminary analysis participants, 180 μL of normal saline was added to 4 microplate wells. Twenty microliters of blood serum was mixed with the normal saline in well 1, making its content volume 200 μL; thus, the dilution factor was 20/200 (a 1:10 ratio of BDNF to saline). Twenty microliters from well 1 was transferred into well 2 and mixed with 180 μL of normal saline, making its content volume 200 μL. The same transfer was performed for wells 3 and 4 to make a 10-fold dilution with each transfer, including a sample duplicate. The sandwich ELISA protocol (Supplemental Materials) was performed on the standardized controls and preliminary serum samples. Supplemental Table S1 shows absorbance of the standard controls (including duplicates) and the preliminary serum samples, quantified in optical density as identified by the microplate reader.
Statistical Analysis
A mixed-model ANOVA was conducted to investigate the differences in serum BDNF concentration before and after exercise with and without BFR. A preset α level of .05 was employed for statistical significance. The SPSS statistical package was used for data analysis. Data are presented as means ± standard deviation unless otherwise stated.
RESULTS
BDNF Response
Twenty participants were enrolled in the present study. Eighteen participants (mean age 34.44 ± 9.97 years) completed the study. One participant was excluded for not adhering to fasting protocol and another because of inability to obtain a postexercise blood sample. Of the 18 successful participants, 9 were male (mean age 38 ± 11.32 years) and 9 were female (mean age 30.89 ± 7.39 years). Table 1 identifies the sex and group allocation of each participant and illustrates the relationship between optical density and serum BDNF concentrations before and after exercise.
Statistical Analysis: Time × Exercise Type (BDNF)
A significant interaction effect for exercise type over time was noted using the Wilks λ test statistic (0.543, F1,16 = 13.477, P < .002, partial η = 0.457). These results suggest that BFRE group had a greater increase in serum levels of BDNF than the control group without BFR. Data met assumptions for normality.
Table 2 reports group means, confidence intervals, and standard deviations of serum concentrations of BDNF before and after exercise for both the experimental and the work-matched control groups. Figure 1 provides a visual representation of the interaction between time and exercise type on BDNF concentration (pg·mL−1). Time 1 is pre-exercise and time 2 is postexercise on the x-axis.
Citation: Journal of Clinical Exercise Physiology 14, 2; 10.31189/2165-6193-14.2.36
Statistical Analysis: Time × Sex (BDNF)
A mixed-model ANOVA was conducted to determine if there was a significant interaction effect of sex on serum concentration levels of BDNF between men and women who performed BFRE. Assumptions for normality and linearity were met. One outlying data point was noted for a male participant; however, this data point was within 2 standard deviations of the group mean. Table 3 reports descriptive statistics demonstrating serum concentrations of BDNF between male and female participants before and after exercise.
Figure 2 provides visual representation of the interaction between sex (male and female) and time.
Citation: Journal of Clinical Exercise Physiology 14, 2; 10.31189/2165-6193-14.2.36
Using the Wilks λ test statistic, a significant interaction effect was found for sex over time related to serum concentrations of BDNF (0.500, F1,8 = 8.010, P < .022, partial η = 0.500).
DISCUSSION
The aim of this study was to explore the acute effects of low-load cycling with BFR on serum concentrations of BDNF. Results of the study support the working hypothesis that serum concentration of BDNF would be significantly higher after exercise in the BFRE group compared with the control group. Additionally, a significant interaction between sex and time was noted, as women in the BFRE group had a greater increase in serum BDNF concentrations compared with men in the BFRE group. If this is reproduced in future studies and different patient populations, it might suggest the importance of adding BFRE in women considering their higher incidence of developing Alzheimer disease (23).
The findings in the present study corroborate the results of Kargaran et al. (16), who found a significant increase in BDNF in older women using BFR while walking on a treadmill and performing dual cognitive tasks. Du et al. (15) demonstrated low-intensity resistance training with BFR increased BDNF significantly compared with low-intensity resistance training without BFR in participants with poststroke depression. Decreased BDNF serum levels correspond to the severity of cognitive impairment (24). BFRE has been demonstrated to be a useful tool in preventing sarcopenia in the older population (1,3). Our findings suggest that adding BFR to low-intensity exercise can increase BDNF compared with low-intensity exercise alone. If further research supports elevations in BDNF in prevention of age-related cognitive decline, BFRE could also be a useful tool for the older population in maintaining brain health.
Study Limitations
The present study examined the effect of BFRE on serum concentration levels of BDNF in healthy adults between the ages of 18 and 50 years. Therefore, caution should be used with attempts to generalize these results to older adults or those with specific medical diagnoses. Additionally, only one bout of BFRE was performed; therefore, the lasting changes in analyte concentration and effects on the nervous system cannot be known from this study. Although the measures were repeated during the same day, the measure was not repeated across multiple days/time points, limiting the generalizability of the results.
Furthermore, the present study examined changes to peripheral levels of BDNF. Although peripheral BDNF concentrations are closely related to concentrations found in the central nervous system (9,10), the precise concentration in the brain could not be accurately measured within the scope of the present study.
The utilization of set occlusion pressures during BFRE is also a potential limitation. Investigators determined set occlusion pressures should be utilized based on protocols established in similar studies (18,19). However, because of variations in underlying soft-tissue pressure and limb circumference among participants, selected cuff pressures may not have uniformly restricted blood flow across all participants. This potential disparity may have caused a differential effect among participants (25).
Suggestions for Implementation and Future Research
BFRE has already demonstrated safe and positive adaptations to muscle performance, including muscle hypertrophy and strength, in older adults (1). Now, evidence is beginning to accumulate supporting the use of BFRE to facilitate metabolic processes that could prevent age-related cognitive decline (15,16). Further research is needed to examine dosing and protocol parameters of BFRE to safely foster gains in BDNF concentrations in older adults. Specifically, a prospective randomized controlled trial using a longitudinal design examining the effects of BFRE in older adults with and without mild cognitive impairment would be invaluable.
In recent years, new evidence has suggested that performing low-load exercise coupled with BFR serves as a potent stimulus for increases in circulating BDNF (12,13). Although these studies support lower extremity exercises in homogenous populations, there have been few empirical studies of the benefits of cardiovascular exercise on the analyte investigated by the present study.
CONCLUSION
The present study found a robust increase in BDNF in the BFRE group compared with the work-matched controls while also detecting a significant difference between sexes with women producing more BDNF than men. Age-related changes in neurocognitive function are leading factors in functional independence and quality of life in older adults. Therefore, further research is needed to determine the effects of BFRE on mitigating the burden associated with disorders of these systems.
Interaction of time and exercise condition on serum concentrations of BDNF between BFRE group and work-matched controls. The y-axis represents the serum BDNF concentration (pg·mL−1). BDNF = brain-derived neurotrophic factor; BFRE = blood flow restriction exercise.
Concentration of BDNF before and after exercise comparing male and female BFRE participants. The y-axis represents the serum BDNF concentration (pg·mL−1). BDNF = brain-derived neurotrophic factor; BFRE = blood flow restriction exercise.
Contributor Notes
Conflicts of Interest and Source of Funding: None.